TECHNICAL FIELD
[0001] Embodiments of the present disclosure relate to a cell scaffold material, a cell
culture support, and a cell culture method.
BACKGROUND ART
[0002] Cell culturing is conducted by inoculating a substrate with the cells, and adding
a medium. One know method used to enhance the adhesion between the substrate and the
cells involves coating the substrate with a cell scaffold material.
[0003] In the field of regenerative medicine, which has expanded significantly in recent
years, research is being conducted into methods in which stem cells typified by iPS
cells and the like are grown by in vitro cell culture, and then used within the body
of animals or humans. In cell culturing, cell proliferation is achieved while successive
cell subcultures are conducted, and it is preferable that cells are grown for which
cell differentiation is suppressed. If a portion of the cells differentiate, cell
proliferation progress is hindered, and particularly in the case of large-scale subcultures,
cell proliferation may sometimes be inhibited. Further, in those cases where cells
having different differentiation stages exist within the culture system, cells from
a specific differentiation stage may sometimes require isolation, but isolating these
cells with a high degree of purity is technically difficult.
[0004] Furthermore, in cell culturing, a protein component can be used as a medium or scaffold
material or the like. If this protein becomes incorporated in the final cultured cell
product, then the protein can sometimes act as an antigen. Furthermore, when the protein
is animal-derived or human-derived, fluctuation between lots can be large, which can
sometimes have an effect on stable culturing of the cells. Accordingly, demands are
increasing for cell culturing in serum-free media. In particular, there is growing
demand for cell scaffold materials that are suitable for stem cell culturing in serum-free
media.
[0005] Up until now, laminin and fibronectin and the like have been investigated as cell
scaffold materials for use in stem cell culturing, but adequate performance has not
been obtainable.
[0006] JP 2018-068192 A discloses that a compound having a structure formed from an aromatic ring and a hydrogen
bonding unit positioned between the blocks of an amphiphilic block copolymer affects
cell proliferation and morphological control. Examples of specific compounds disclosed
in
JP 2018-068192 A include compounds having an N-(4-(ureidomethyl)benzyl)benzamide structure between
the blocks of a block copolymer of polyethylene glycol (PEG) and poly-L-lactic acid
(PLLA), poly-ε-caprolactone (PCL) or trimethylene carbonate (PTMC).
[0007] JP 2018-068192 A proposes the use of these compounds as cell spreading and growth control agents by
addition to the culture medium.
SUMMARY OF INVENTION
PROBLEMS INVENTION AIMS TO SOLVE
[0008] JP 2018-068192 A only discloses applications of these compounds as cell spreading and growth control
agents that are added to the culture medium, and little investigation was conducted
into cell scaffold materials.
[0009] Cell scaffold materials require favorable adhesion between the substrate and cells,
while having no adverse effects on cell proliferation. Moreover, in cell culturing
using a cell scaffold material, it is sometimes desirable to promote cell proliferation
while suppressing differentiation.
[0010] The present disclosure has the objects of providing a cell scaffold material, a cell
culture support and a cell culture method that suppress cell differentiation and promote
cell proliferation.
MEANS FOR SOLUTION OF THE PROBLEMS
[0011] Specific aspects for achieving the above objects are as described below.
- [1] A cell scaffold material comprising a copolymer having a polylactic acid structural
unit (A) and a polycarbonate structural unit (B).
- [2] The cell scaffold material according to [1], wherein the polycarbonate structural
unit (B) includes at least one unit having an alkoxyalkyloxycarbonyl group as a substituent.
- [3] The cell scaffold material according to [1] or [2], wherein the polycarbonate
structural unit (B) includes at least one unit having a methoxyethyloxycarbonyl group
as a substituent.
- [4] The cell scaffold material according to [1], wherein the polycarbonate structural
unit (B) includes at least one unit represented by general formula (I) shown below.
![](https://data.epo.org/publication-server/image?imagePath=2022/18/DOC/EPNWA1/EP20832378NWA1/imgb0001)
(In general formula (I), X represents a hydrogen atom or an alkyl group having not
more than 5 carbon atoms.)
- [5] The cell scaffold material according to [4], wherein X in general formula (I)
is a methyl group.
- [6] The cell scaffold material according to any one of [1] to [5], wherein the copolymer
is a block copolymer in which one terminal or both terminals have the polylactic acid
structural unit (A).
- [7] The cell scaffold material according to [6], wherein the copolymer is an AB-type,
ABA-type or ABAB-type block copolymer.
- [8] The cell scaffold material according to [7], wherein the copolymer is an ABA-type
block copolymer.
- [9] The cell scaffold material according to any one of [1] to [8], wherein the polylactic
acid structural unit (A) is a poly-D-lactic acid structural unit or a poly-L-lactic
acid structural unit.
- [10] A cell culture support comprising the cell scaffold material according to any
one of [1] to [9], and a substrate.
- [11] A cell culture method that comprises: preparing the cell scaffold material according
to any one of [1] to [9], placing the cell scaffold material in a culture system,
and culturing cells in the presence of the cell scaffold material.
- [12] The cell culture method according to [11], wherein the culture system comprises
a serum-free medium.
EFFECTS OF THE INVENTION
[0012] The present disclosure provides a cell scaffold material, a cell culture support
and a cell culture method that suppress cell differentiation and promote cell proliferation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013]
FIG. 1 is a graph illustrating the fluorescent intensity of fluorescent-labeled BSA
when supports coated with various polymers are used.
FIG. 2 is a series of fluorescence difference microscope photographs of normal human
fibroblasts cultured using supports coated with various polymers, the photographs
showing the states (a) one hour after the start of cell culturing, (b) one day after
the start of cell culturing, (c) two days after the start of cell culturing, (d) three
days after the start of cell culturing, and (e) seven days after the start of cell
culturing.
FIG. 3 is a graph showing the number of cells relative to the cell culture time of
normal human fibroblasts cultured using supports coated with various polymers.
FIG. 4 is a graph showing the FGF-2 concentration relative to the cell culture time
of normal human fibroblasts cultured using supports coated with various polymers.
EMBODIMENTS FOR CARRYING OUT THE INVENTION
[0014] Embodiments of the present disclosure are described below, but the present disclosure
is not limited to the following embodiments.
[0015] A cell scaffold material according to one of embodiments comprises a copolymer having
a polylactic acid structural unit (A) and a polycarbonate structural unit (B).
[0016] This embodiment can suppress cell differentiation and promote cell proliferation.
[0017] To explain further, the polylactic acid structural unit and the polycarbonate structural
unit that constitute the cell scaffold material according to the present disclosure
both have favorable adhesion to cells and high biocompatibility. On the other hand,
the polylactic acid structural unit has a hydrophobic property, whereas the polycarbonate
structural unit, despite being water-insoluble, has a hydrophilic property compared
with the polylactic acid structural unit. Based on these properties, due to the respective
characteristics of the structural units, the copolymer has a tendency to self-organize
while maintaining adhesiveness to the cells. It is surmised that when the copolymer
is used as a cell scaffold material, this structural characteristic has the effects
of suppressing cell differentiation and promoting cell proliferation. However, the
present disclosure is not constrained by this theory.
[0018] The cell scaffold material according to one of embodiments preferably comprises a
copolymer having a polylactic acid structural unit (A) and a polycarbonate structural
unit (B). This copolymer is preferably a block copolymer of the structural unit (A)
and the structural unit (B).
[0019] In the copolymer according to one of embodiments, the polylactic acid structural
unit (A) means a structural unit formed from a polylactic acid structure, and may
be a structural unit containing a plurality of lactic acid units.
[0020] Examples of the polylactic acid structural unit (A) include poly-D-lactic acid structural
units, poly-L-lactic acid structural units, and poly-DL-lactic acid structural units.
The polylactic acid structural unit (A) is preferably a poly-D-lactic acid structural
unit or a poly-L-lactic acid structural unit, and is more preferably a poly-D-lactic
acid structural unit. In those cases where the copolymer contains two or more polylactic
acid structural units (A), the polylactic acid structural units (A) contained in a
single molecule of the copolymer may all be the same, or some or all of the structural
units (A) may be different, but all of the structural units (A) are preferably the
same.
[0021] In the copolymer according to one of embodiments, the polycarbonate structural unit
(B) means a structural unit formed from a polycarbonate structure, and may be a structural
unit containing a plurality of carbonate units.
[0022] The polycarbonate structural unit is a polymer structural unit having a carbonate
linkage in the main chain, and may be either an aliphatic polycarbonate structural
unit or an aromatic polycarbonate structural unit, but is preferably an aliphatic
polycarbonate structural unit.
[0023] Examples of aliphatic polycarbonate structural units include copolymer structural
units such as polyethylene carbonate structural units, polypropylene carbonate structural
units and polytrimethylene carbonate structural units, as well as derivative structural
units in which side chains have been introduced into these types of structural units.
Examples of aromatic polycarbonate structural units include polyaryl carbonate structural
units and the like, and derivatives of these structural units. Examples of polyaryl
carbonate structural units include polyphenyl carbonate structural units and the like.
[0024] In those cases where the copolymer contains two or more polycarbonate structural
units (B), the polycarbonate structural units (B) contained in a single molecule of
the copolymer may all be the same, or some or all of the structural units (B) may
be different, but all of the structural units (B) are preferably the same.
[0025] The carbonate units that constitute the polycarbonate structural unit (B) may have
a substituent. The polycarbonate structural unit (B) preferably has at least one carbonate
unit having an alkoxyalkyloxycarbonyl group as a substituent. The carbonate units
having this substituent preferably constitute one or more of the carbonate units in
the polycarbonate structural unit (B), or may represent 80 mol% or more of all of
the carbonate units that constitute the polycarbonate structural unit (B), or all
of the carbonate units may have this substituent.
[0026] The alkoxyalkyloxycarbonyl group is preferably introduced by direct bonding to a
carbon atom of a carbonate linkage in the main chain.
[0027] Further, in the alkoxyalkyloxycarbonyl group, the alkoxy group may be linear or branched,
is preferably an alkoxy group of not more than 5 carbon atoms, more preferably an
alkoxy group of not more than 3 carbon atoms, and even more preferably an ethoxy group
or methoxy group, with a methoxy group being particularly preferred.
[0028] Furthermore, in the alkoxyalkyloxycarbonyl group, the alkylene group interposed
between the alkoxy group and the carbonyl group may be linear or branched, preferably
has not more than 5 carbon atoms and more preferably not more than 3 carbon atoms,
and is even more preferably a propylene group or ethylene group, with an ethylene
group being particularly preferred.
[0029] Specific examples of the alkoxyalkyloxycarbonyl group include a methoxyethyloxycarbonyl
group, methoxypropyloxycarbonyl group, ethoxyethyloxycarbonyl group and ethoxypropyloxycarbonyl
group, and a methoxyethyloxycarbonyl group is particularly preferred.
[0030] As a result of the copolymer having an alkoxyalkyloxycarbonyl group such as a methoxyethyloxycarbonyl
group as a substituent of the polycarbonate structural unit (B), the adhesion to cells
can be further enhanced, and cell differentiation during cell proliferation can be
better suppressed. It is thought that by incorporating this substituent, the copolymer
can more easily adopt a layer structure containing a water molecule between the molecular
chains of water-insoluble structural units, with cells held stably in the polycarbonate
structural unit (B) portion, meaning cell proliferation is better promoted and cell
differentiation is better suppressed, although the present disclosure is not constrained
by this theory.
[0031] In one preferred example, the polycarbonate structural unit (B) preferably contains
at least one carbonate unit represented by general formula (II) shown below.
![](https://data.epo.org/publication-server/image?imagePath=2022/18/DOC/EPNWA1/EP20832378NWA1/imgb0002)
[0032] In general formula (II), M represents a hydrogen atom, a linear or branched alkyl
group of not more than 5 carbon atoms, or a group represented by -L-Z, m and m' each
independently represent an integer of 0 to 5, provided that m+m' = 1 to 7, Y is a
group represented by -L-Z, L represents an alkylene group, ether linkage, ester linkage,
single bond, -C(=O)-, or a divalent group having a combination of these moieties,
and Z represents a chain-like ether group, cyclic ether group, group having an acetal
structure, alkoxy group, alkoxyalkyl group, or a monovalent group having a combination
of these groups.
[0033] The polycarbonate structural unit (B) may be a unit in which a plurality of carbonate
units represented by general formula (II) are polymerized. In this case, the degree
of polymerization of the units represented by general formula (II) may be, for example,
within a range from 2 to 2,000.
[0034] The polycarbonate structural unit may contain at least one, or may contain two or
more, of the carbonate units represented by general formula (II), and 80 mol% or more
of all of the carbonate units included in the polycarbonate structural unit are preferably
units represented by general formula (II). Moreover, all of the carbonate units included
in the polycarbonate structural unit may be units represented by general formula (II).
[0035] Further, in those cases where the polycarbonate structural unit includes a plurality
of units represented by general formula (II), the plurality of units represented by
general formula (II) may all be the same, or some or all of the units may be different.
[0036] In general formula (1), M may represent a hydrogen atom, a linear or branched alkyl
group of not more than 5 carbon atoms, preferably not more than 3 carbon atoms, and
more preferably not more than 2 carbon atoms, or a group represented by -L-Z. Specific
examples include a hydrogen atom, methyl group, ethyl group, propyl group, isopropyl
group, butyl group, isobutyl group, tert-butyl group, sec-butyl group, pentyl group,
isopentyl group, sec-pentyl group, and tert-pentyl group. Further, the group represented
by -L-Z is as described below in relation to Y. Of the various possibilities, M is
preferably a methyl group or ethyl group, and is more preferably a methyl group.
[0037] Further, m and m' each independently represent an integer of 0 to 5, and preferably
satisfy m+m' = 1 to 7. Moreover, it is preferable that m and m' satisfy m+m' = 1 to
4, and more preferable that m=m'=1. In those cases where m=m'=1, the main chain of
the polycarbonate structural unit has a polytrimethylene carbonate skeleton.
[0038] Furthermore, Y is a group represented by -L-Z, wherein L and Z are as described
below.
[0039] L is a linker between the main chain and Z, and may be an alkylene group, ether linkage,
ester linkage, single bond, -C(=O)-, or a divalent group having a combination of these
moieties, and among these possibilities, is preferably an ether linkage, ester linkage,
single bond, -C(=O)-, or a divalent group having a combination of these moieties,
and is more preferably an ester linkage or a divalent group having -C(=O)-.
[0040] Z may be a chain-like ether group, cyclic ether group, group having an acetal structure,
alkoxy group, alkoxyalkyl group, or a monovalent group having a combination of these
groups, and among these possibilities, is preferably a chain-like ether group or an
alkoxyalkyl group.
[0041] The chain-like ether group preferably has, for example, a structure composed of an
alkylene glycol structure such as ethylene glycol or propylene glycol, or a polymer
thereof.
[0042] Specific examples of Z groups of general formula (III) shown below in which R is
an ethylene group or a propylene group, R' is a hydrogen atom or a linear or branched
alkyl group of not more than 5 carbon atoms, and n represents an integer of 1 to 30.
-(R-O)n-R' (III)
[0043] In general formula (III), R is preferably an ethylene group. Further R' is preferably
a methyl group or an ethyl group, and is more preferably a methyl group. Furthermore,
n is preferably an integer of 1 to 5, and more preferably 1 or 2.
[0044] It is more preferable that Z is an alkoxyalkyl group, for example, a methoxyethyl
group, methoxypropyl group, ethoxyethyl group, ethoxypropyl group, propyloxyethyl
group or propyloxypropyl group, and of these, a methoxyethyl group is preferred.
[0045] Specific examples of the group represented by -L-Z include -OCH3, -OCH
2CH
3, - OCH
2CH
2CH
3, -CH
2OCH
3, -CH
2OCH
2CH
3, -CH
2OCH
2CH
2CH
3, -CH
2CH
2OCH
3, - CH
2CH
2OCH
2CH
3, -CH
2CH
2OCH
2CH
2CH
3, -CH
2OCH
2CH
2OCH
3, - CH
2OCH
2CH
2OCH
2CH
3, -CH
2OCH
2CH
2OCH
2CH
2CH
3, -CH
2CH
2OCH
2CH
2OCH
3, - CH
2CH
2OCH
2CH
2OCH
2CH
3, -CH
2CH
2OCH
2CH
2OCH
2CH
2CH
3, -C(=O)OCH
3, - C(=O)OCH
2CH
3, -C(=O)OCH
2CH
2CH
3, -C(=O)OCH
2CH
2OCH
3, - C(=O)OCH
2CH
2CH
2OCH
3, -C(=O)OCH
2CH
2OCH
2CH
3, - C(=O)OCH
2CH
2CH
2OCH
2CH
3, -C(=O)OCH
2CH
2OCH
2CH
2CH
3, and - C(=O)OCH
2CH
2CH
2OCH
2CH
2CH
3. Among these groups, -C(=O)OCH
3, - C(=O)OCH
2CH
3, -C(=O)OCH
2CH
2CH
3, -C(=O)OCH
2CH
2OCH
3, - C(=O)OCH
2CH
2CH
2OCH
3, -C(=O)OCH
2CH
2OCH
2CH
3, - C(=O)OCH
2CH
2CH
2OCH
2CH
3, -C(=O)OCH
2CH
2OCH
2CH
2CH
3 or - C(=O)OCH
2CH
2CH
2OCH
2CH
2CH
3 is preferred. Further, -C(=O)OCH
2CH
2OCH
3, - C(=O)OCH
2CH
2CH
2OCH
3, -C(=O)OCH
2CH
2OCH
2CH
3, - C(=O)OCH
2CH
2CH
2OCH
2CH
3, -C(=O)OCH
2CH
2OCH
2CH
2CH
3 or - C(=O)OCH
2CH
2CH
2OCH
2CH
2CH
3 is more preferred. The group -C(=O)OCH
2CH
2OCH
3 is particularly preferred.
[0046] In one specific preferred example, the polycarbonate structural unit (B) preferably
contains at least one carbonate unit represented by general formula (I) shown below.
![](https://data.epo.org/publication-server/image?imagePath=2022/18/DOC/EPNWA1/EP20832378NWA1/imgb0003)
[0047] In general formula (I), X represents a hydrogen atom or an alkyl group of not more
than 5 carbon atoms.
[0048] The alkyl group of not more than 5 carbon atoms for X may be a linear or branched
group. Further, X is preferably a methyl group or ethyl group, and is more preferably
a methyl group.
[0049] The polycarbonate structural unit (B) may be a unit in which a plurality of carbonate
units represented by general formula (I) are polymerized. In this case, the degree
of polymerization of the units represented by general formula (I) may be, for example,
within a range from 2 to 2,000.
[0050] The polycarbonate structural unit may contain at least one, or may contain two or
more, of the carbonate units represented by general formula (I), and 80 mol% or more
of all of the carbonate units included in the polycarbonate structural unit are preferably
units represented by general formula (I). Moreover, all of the carbonate units included
in the polycarbonate structural unit may be units represented by general formula (I).
[0051] Further, in those cases where the polycarbonate structural unit includes a plurality
of units represented by general formula (I), the plurality of units represented by
general formula (I) may all be the same, or some or all of the units may be different.
[0052] The copolymer according to one of embodiments may be a block copolymer containing
the polylactic acid structural unit (A) and the polycarbonate structural unit (B).
In this case, the block structure of the block copolymer preferably has at least one
terminal formed from the polylactic acid structural unit (A). In other words, the
block structure of the block copolymer preferably has one terminal or both terminals
formed from the polylactic acid structural unit (A). Examples of the block structure
include AB-type, ABA-type and ABAB-type structures, and an ABA-type structure is preferred.
[0053] In the block structure of the block copolymer, by ensuring that one terminal or both
terminals are formed from the polylactic acid structural unit (A), because the polylactic
acid structural unit (A) exhibits favorable adhesion to the substrate, the cell scaffold
material can be adhered more stably to the substrate.
[0054] Moreover, it is thought by incorporating the polylactic acid structural unit (A)
at both terminals, such as in an ABA-type structure, the block copolymer can adhere
to the substrate with the polylactic acid structural units (A) adhered to the substrate
at both terminals of the block copolymer, and the intermediate polycarbonate structural
unit (B) floating above the substrate. With this configuration, cells can be captured
by the floating polycarbonate structural unit (B) portion, while the adhesion of the
cells to the substrate is enhanced.
[0055] One example of the block copolymer is a copolymer represented by general formula
(IV) shown below. More specifically, a copolymer represented by general formula (IV)
shown below is preferred.
![](https://data.epo.org/publication-server/image?imagePath=2022/18/DOC/EPNWA1/EP20832378NWA1/imgb0005)
[0056] In general formula (IV), X is the same as described above for general formula (I),
and therefore description here is omitted. In general formula (IV), a, b, c and d
are numerical values that may be determined appropriately in accordance with the molecular
weight of the copolymer, and these values may all be the same, or some or all of the
values may be different. For example, it is preferable that one or both of a=d and
b=c are satisfied, and more preferably that a=d and b=c.
[0057] In general formula (V), a, b, c and d are the same as described above for general
formula (IV), and therefore descriptions here are omitted.
[0058] The number average molecular weight (Mn) of one molecule of the copolymer according
to one of embodiments is preferably within a range from 5,000 to 100,000, more preferably
from 10,000 to 80,000, even more preferably from 12,500 to 70,000, and particularly
preferably from 15,000 to 50,000.
[0059] The molecular weight distribution (Mw/Mn) for one molecule of the copolymer according
to one of embodiments is not particularly limited, but is, for example, typically
not more than 2.0, and is preferably not more than 1.5, and more preferably 1.2 or
less.
[0060] The number average molecular weight (Mn) of the polylactic acid structural unit (A)
is preferably within a range from 1,000 to 50,000, more preferably from 3,000 to 40,000,
even more preferably from 4,000 to 35,000, and still more preferably from 5,000 to
30,000.
[0061] Further, the molecular weight distribution (Mw/Mn) for the polylactic acid structural
unit (A) is not particularly limited, but is preferably within a range from 1.0 to
10, more preferably from 1.0 to 8, and even more preferably from 1.05 to 5.
[0062] The number average molecular weight (Mn) of the polycarbonate structural unit (B)
is preferably within a range from 2,000 to 50,000, more preferably from 5,000 to 40,000,
even more preferably from 6,000 to 35,000, and still more preferably from 7,000 to
20,000.
[0063] Further, the molecular weight distribution (Mw/Mn) for the polycarbonate structural
unit (B) is not particularly limited, but is preferably within a range from 1.0 to
10, more preferably from 1.0 to 8, and even more preferably from 1.05 to 5.
[0064] Here, the number average molecular weight of the polylactic acid structural unit
(A) refers to the number average molecular weight of one block, and in those cases
where the block copolymer includes two or more blocks of the polylactic acid structural
unit (A), each block preferably satisfies the above range. This also applies to the
polycarbonate structural unit (B).
[0065] Further, the number average molecular weight (Mn) and the weight average molecular
weight (Mw) can be measured using gel permeation chromatography (GPC) calibrated against
standard polystyrenes. Furthermore, the molecular weight distribution can be determined
from the ratio (Mw/Mn) of the weight average molecular weight (Mw) relative to the
number average molecular weight (Mn).
[0066] In the copolymer according to one of embodiments, it is preferable that all of the
units that constitute the polylactic acid structural unit (A) are lactic acid units.
Further, in the copolymer according to one of embodiments, it is preferable that all
of the units that constitute the polycarbonate structural unit (B) are carbonate units.
[0067] In the copolymer according to one of embodiments, the total number of monomer units
constituting the polylactic acid structural unit (A), relative to the total number
of monomer units constituting the entire copolymer, is preferably at least 10 mol%,
more preferably at least 30 mol%, and even more preferably 40 mol% or greater.
[0068] Further, in the copolymer according to another embodiment, the total number of monomer
units constituting the polylactic acid structural unit (A), relative to the total
number of monomer units constituting the entire copolymer, is preferably not more
than 90 mol%, more preferably not more than 70 mol%, and even more preferably 60 mol%
or less.
[0069] The total number of monomer units constituting the polylactic acid structural unit
(A), relative to the total number of monomer units constituting the entire copolymer,
is preferably within a range from 10 to 90 mol%, more preferably from 30 to 70 mol%,
and even more preferably from 40 to 60 mol%.
[0070] In the copolymer according to one of embodiments, the total number of monomer units
constituting the polycarbonate structural unit (B), relative to the total number of
monomer units constituting the entire copolymer, is preferably at least 10 mol%, more
preferably at least 30 mol%, and even more preferably 40 mol% or greater.
[0071] Further, in the copolymer according to another embodiment, the total number of monomer
units constituting the polycarbonate structural unit (B), relative to the total number
of monomer units constituting the entire copolymer, is preferably not more than 90
mol%, more preferably not more than 70 mol%, and even more preferably 60 mol% or less.
[0072] The total number of monomer units constituting the polycarbonate structural unit
(B), relative to the total number of monomer units constituting the entire copolymer,
is preferably within a range from 10 to 90 mol%, more preferably from 30 to 70 mol%,
and even more preferably from 40 to 60 mol%.
[0073] In the copolymer according to one of embodiments, the molar ratio between the total
number of monomer units constituting the polylactic acid structural unit (A) and the
total number of monomer units constituting the polycarbonate structural unit (B) is
preferably within a range from 10:90 to 90:10, more preferably from 30:70 to 70:30,
and even more preferably from 40:50 to 50:40.
[0074] In the copolymer according to one of embodiments, the combined total of the total
number of monomer units constituting the polylactic acid structural unit (A) and the
total number of monomer units constituting the polycarbonate structural unit (B),
relative to the total number of monomer units constituting the entire copolymer, is
preferably at least 80 mol%, and more preferably 90 mol% or greater. Moreover, the
copolymer according to one of embodiments may be composed only of the polylactic acid
structural unit (A) and the polycarbonate structural unit (B).
[0075] In those cases where the copolymer according to one of embodiments is a block copolymer,
wherein the block copolymer contains two or more blocks of the polylactic acid structural
unit (A), it is preferable that the total number of monomer units constituting the
two or more blocks of the polylactic acid structural unit (A) satisfies the range
described above. This also applies to the polycarbonate structural unit (B).
[0076] One example of a method for synthesizing the copolymer according to one of embodiments
is described below. The copolymer according to one of embodiments as described above
is not limited to copolymers obtained using the following synthesis method.
[0077] The copolymer can be synthesized by preparing a polylactic acid and a polycarbonate,
and then bonding the polylactic acid and the polycarbonate together.
[0078] Further, the copolymer can also be synthesized by preparing a polycarbonate, and
then polymerizing and bonding a lactic acid, lactic acid lactide or combination thereof
as a monomer at one terminal or both terminals of the polycarbonate.
[0079] Furthermore, the copolymer can also be synthesized by preparing a polylactic acid,
and then polymerizing and bonding a carbonate monomer at one terminal or both terminals
of the polylactic acid.
[0080] Moreover, the copolymer can also be synthesized by using a lactic acid and a carbonate,
and performing either a random polymerization, or a block copolymerization using a
polymerization reagent.
[0081] For the polylactic acid, poly-D-lactic acid, poly-L-lactic acid or poly-DL-lactic
acid or the like may be used individually, or a combination of these compounds may
be used, but the use of either poly-D-lactic acid or poly-L-lactic acid as a single
component is preferred, and the use of poly-D-lactic acid as a single component is
more preferred.
[0082] The number average molecular weight of the polylactic acid is preferably set accordingly
in accordance with the target molecular weight of the structural unit (A) to be introduced
into the block copolymer.
[0083] Examples of the monomer component that constitutes the polylactic acid include lactic
acids, lactic acid lactides, and combinations of lactic acids and lactic acid lactides.
[0084] D-lactic acid, L-lactic acid or DL-lactic acid or the like may be used individually
as the lactic acid monomer component that constitutes the polylactic acid, or a combination
of these lactic acid compounds may be used, but the use of either D-lactic acid or
L-lactic acid as a single component is preferred, and the use of D-lactic acid as
a single component is more preferred.
[0085] D-lactic acid lactide, L-lactic acid lactide or DL-lactic acid lactide or the like
may be used individually as the lactic acid lactide monomer component, or a combination
of these lactic acid lactide compounds may be used, but the use of either D-lactic
acid lactide or L-lactic acid lactide as a single component is preferred, and the
use of D-lactic acid lactide as a single component is more preferred.
[0086] A combination of a lactic acid and a lactic acid lactide may also be used as the
monomer component, but each compound may also be used individually.
[0087] An aliphatic polycarbonate or an aromatic polycarbonate may be used alone as the
polycarbonate, or a combination of these types may be used.
[0088] A polycarbonate derivative containing at least one carbonate unit having an alkoxyalkyloxycarbonyl
group as a substituent can be used favorably as the polycarbonate, and it is even
more preferable that this substituent is a methoxyethyloxycarbonyl group.
[0089] Further, the use of a polycarbonate derivative containing at least one carbonate
unit represented by general formula (II) as the polycarbonate is preferred. Moreover,
the use of a polycarbonate derivative containing at least one carbonate unit represented
by general formula (I) is also preferred. The carbonate unit represented by general
formula (II) is preferably a carbonate unit having an alkoxyalkyloxycarbonyl group
as a substituent. Further, the carbonate unit represented by general formula (I) is
preferably a carbonate unit having a methoxyethyloxycarbonyl group as a substituent.
[0090] The number average molecular weight of the polycarbonate is preferably set accordingly
in accordance with the target molecular weight of the polycarbonate structural unit
(B) to be introduced into the block copolymer.
[0091] The carbonate monomer component that constitutes the polycarbonate is preferably
an aliphatic carbonate, and examples include ethylene carbonate, propylene carbonate,
and trimethylene carbonate and the like, as well as derivatives and the like of these
compounds. These compounds may be used individually, or combinations two or more compounds
may be used. A carbonate having a structure represented by general formula (II) or
a cyclic carbonate that represents a cyclic derivative of such a compound is particularly
preferred. Further, trimethylene carbonate derivatives described below may also be
used.
[0092] Copolymerization of the polylactic acid or polylactic acid monomer component and
the polycarbonate or polycarbonate monomer component is preferably conducted by solution
polymerization, and if necessary, a polymerization initiator and/or polymerization
catalyst or the like may be added to the synthesis system.
[0093] Examples of the polymerization solvent include dichloromethane, chloroform, diethyl
ether, tetrahydrofuran (THF) and toluene.
[0094] Examples of the polymerization initiator include alcohol-based polymerization initiators
such as 1-pyrenebutanol, lauryl alcohol, decanol and stearyl alcohol, and cyclic amine
polymerization initiators such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), dimethylaminopyridine
(DMAP), triethylenediamine (DABCO), (+)-sparteine, and (-)-sparteine.
[0095] Examples of the polymerization catalyst include difunctional thiourea compounds such
as 1-(3,5-bis(trifluoromethyl)phenyl)-3-cyclohexyl-2-thiourea (TU).
[0096] The polymerization is preferably conducted at a temperature range from room temperature
to any temperature that does not affect the synthesis system, and more specifically
at a temperature within a range from room temperature to 50°C, either in an open atmosphere
or under an inert atmosphere, and preferably under an inert atmosphere such as a nitrogen
atmosphere, and is preferably conducted for a period from 1 minute to 12 hours, and
more preferably a period from 30 minutes to 6 hours.
[0097] Completion of the polymerization reaction can be confirmed by whether or not the
monomer components and the like are still present in the reaction system, and can
be confirmed by methods such as
1H-NMR and TLC. Once the polymerization reaction has progresses sufficiently, the polymerization
reaction may be stopped by adding a reaction terminator. Examples of the reaction
terminator include acetic acid, hydrochloric acid, sulfuric acid and benzoic acid.
[0098] A method for synthesizing a polycarbonate derivative having an alkoxyalkyloxycarbonyl
group as a substituent is described below. This polycarbonate derivative can be obtained
by ring-opening polymerization of a monomer having a functional group introduced as
a side chain into a cyclic carbonate skeleton.
[0099] As a more specific example, a method for synthesizing a polycarbonate derivative
containing a carbonate unit represented by general formula (II) is described below.
[0100] For example, in order to polymerize a polytrimethylene carbonate derivative having
a unit represented by general formula (II) in which m=m'=1, a trimethylene carbonate
derivative having a group represented by M and a group represented by Y introduced
on a carbon atom of the trimethylene carbonate may be used as a monomer. M and Y are
as described above in relation to general formula (II).
[0101] Specific examples of the trimethylene carbonate derivative include 5-methyl-5-(2-methoxyethyloxycarbonyl)-1,3-dioxan-2-one,
5-methyl-5-(2-ethoxyethyloxycarbonyl)-1,3-dioxan-2-one, 5-methyl-5-[2-(2-methoxyethoxy)ethyloxycarbonyl]-1,3-dioxan-2-one,
4-methyl-4-(2-methoxyethyloxycarbonyl)-1,3 -dioxan-2-one, 4-methyl-4-(2-ethoxyethyloxycarbonyl)-1,3-dioxan-2-one,
and 4-methyl-4-[2-(2-methoxyethoxy)ethyloxycarbonyl]-1,3-dioxan-2-one.
[0102] In the synthesis of a polycarbonate derivative having a unit represented by general
formula (II), the ring-opening polymerization of the monomer having a functional group
introduced as a side chain into the cyclic carbonate skeleton can be conducted by
any of various methods without any particular limitations. For example, the ring-opening
polymerization may be conducted by a cationic polymerization reaction or an anionic
polymerization reaction using any of various polymerization initiators, and if necessary,
a polymerization catalyst or the like may also be used. There are no particular limitations
on the polymerization solvent, the polymerization initiator and the polymerization
catalyst, and the types of compounds described above in relation to synthesis of the
above copolymer may be used.
[0103] More specifically, synthesis may be conducted in accordance with the method disclosed
in
JP 2014-161675 A.
[0104] The cell scaffold material according to one of embodiments may contain the copolymer
according to the embodiment described above as the only component.
[0105] Further, the cell scaffold material according to one of embodiments, may also be
provided in the form of a cell scaffold material composition containing an added solvent.
Examples of the solvent include water, organic solvents, and mixed solvents thereof.
The water may include not only water used as a solvent, but also intermediate water
incorporated in the copolymer according to one of embodiments. Examples of the organic
solvents include dichloromethane and methanol. The cell scaffold material composition
may also contain, as necessary, medium additives described below and other additive
components and the like.
[0106] Further, one of embodiments is able to provide a cell culture support containing
the cell scaffold material according to one of embodiments described above and a substrate.
[0107] The cell culture support according to one of embodiments has the effects of suppressing
cell differentiation and promoting cell proliferation, and can therefore be used favorably
for growing cells of a specific differentiation stage by culturing.
[0108] There are no particular limitations on the shape of the substrate included in the
support, and one or more shapes selected from the group consisting of flat plates,
curved plates, spheres and blocks may be used.
[0109] Specifically, any of the materials typically used as cell culture substrates may
be used as the substrate, and examples of substrates that may be used include dishes,
well plates such as flat-bottom well plates and round-bottom well plates; cell containers
such as Petri dishes; microbeads, microcarriers and three-dimensional blocks; and
cell sheets and the like.
[0110] Although there are no particular limitations on the material of the substrate, a
material that displays no cell toxicity is preferred. Examples of the material include
resins including ester-based resins such as polyethylene terephthalate, (meth)acrylic-based
resins, epoxy-based resins, urethane-based resins, styrene-based resins, thiol-based
resins and silicone-based resins; metals such as pure nickel, titanium, platinum,
gold, tungsten, rhenium, palladium, rhodium and ruthenium; alloys such as stainless
steel, titanium/nickel, nitinol, cobalt/chromium and platinum/iridium alloys; and
glass and ceramics and the like.
[0111] Furthermore, the substrate that has undergone cell culturing may be used in regenerative
medicine where the substrate is transplanted into a living body. Examples of substrates
that are suitable for regenerative medicine include biocompatible materials such as
silicone, polyether block amides (PEBAX), polyurethane, silicone-polyurethane copolymers,
ceramics, collagen, hydroxyapatite, nylon, polyethylene terephthalate, ultrahigh molecular
weight polyethylenes such as Gore-Tex (a registered trademark), polyvinyl chloride,
and other tissue-derived biomaterials; polylactide (PLA), polyglycolide (PGA), polycaprolactone
(PCL) and copolymers of these polymers, PHB-PHV-based poly(alkanoic acids), polyesters,
natural polymers such as starch, cellulose and chitosan, and derivatives of the above
materials.
[0112] The cell culture support can be obtained by applying the cell scaffold material to
the substrate.
[0113] For example, the cell scaffold material may be dissolved in an organic solvent, and
the resulting liquid composition then applied to the substrate.
[0114] Examples of solvents that may be used include dichloromethane and methanol and the
like. Further, in addition to the cell scaffold material, the liquid composition may
also contain, medium additives described below and other additive components and the
like. The amount of the cell scaffold material relative to the total mass of the liquid
composition is preferably within a range from 0.05 to 10% by mass, and more preferably
from 0.1 to 1% by mass.
[0115] There are no particular limitations on the method used for applying the liquid composition
to the substrate, and for example, a method using spin coating may be used.
[0116] The amount of the cell scaffold material applied to the substrate may be adjusted
appropriately in accordance with factors such as the type of substrate, the type of
cell scaffold material, the type of cell used as the culture target, and the application
method. The amount of the cell scaffold material applied to the substrate, expressed
as a solid fraction amount per unit of surface area, may be set, for example, to a
value within a range from 0.05 µg/mm
2 to 500 µg/mm
2. In those cases where an application method employing a spin coater is used, the
solid fraction amount per unit of surface area, may be set, for example, to a value
within a range from 0.01 µg/mm
2 to 10 µg/mm
2, to a value within a range from 0.05 µg/mm
2 to 5 µg/mm
2, or a value within a range from 0.1 µg/mm
2 to 3.0 µg/mm
2.
[0117] Before applying the cell scaffold material to the substrate, the substrate may be
treated with another polymer besides the copolymer according to one of embodiments
as described above. Examples of the other polymer include (meth)acrylic-based resins
and the like. The other polymer is preferably combined with a suitable solvent, and
applied to the substrate in liquid form. By interposing another polymer between the
substrate and the cell scaffold material, adhesion of the scaffold material to the
substrate can be enhanced.
[0118] The cell culture support according to one of embodiments may be placed in a culture
system of the cells that are targeted for proliferation. Specifically, the cell culture
support may be first placed in the medium, and the medium then inoculated with the
target cells, or the cell culture support may be supplied to a medium in which the
target cells already exist. As a result, the target cells and the cell culture support
make contact, and the target cells adhere to the cell culture support and grow. Accordingly,
by using the cell culture support according to one of embodiments, differentiation
of the target cells can be suppressed and cell proliferation can be promoted.
[0119] A cell culture method according to one of embodiments is described below.
[0120] The cell culture method according to one of embodiments may include preparing a cell
scaffold material, placing the cell scaffold material in a culture system, and culturing
the cells in the presence of the cell scaffold material.
[0121] The cell scaffold material according to one of embodiments described above can be
used as the cell scaffold material. As a result, cell differentiation can be suppressed,
cell proliferation can be promoted, and a large number of undifferentiated cells can
be manufactured.
[0122] In the step of preparing the cell scaffold material, the cell scaffold material according
to one of embodiments described above is prepared. At this time, the cell scaffold
material may be provided in the form of a composition containing the cell scaffold
material as one component, or may be provided in the form of a cell culture support
containing the cell scaffold material and a substrate.
[0123] In the step of placing the cell scaffold material in the culture system, a placement
method is selected appropriately in accordance with the form in which the cell scaffold
material is provided, and the cell scaffold material is placed in the culture system.
For example, the cell scaffold material may be placed in the culture system prior
to cell inoculation in the form of a cell scaffold material composition or a cell
culture support containing a plate-shaped substrate. In an alternative method, the
culture system may first be inoculated with the target cells, and the cell scaffold
material then subsequently placed in the culture system as a cell scaffold material
composition or a cell culture support in the form of microbeads.
[0124] In the step of conducting culturing of the cells, culturing of the target cells is
conducted in the presence of the cell scaffold material.
[0125] Examples of cells that may be used as the culture target include primary culture
cells, cultured cell lines, and recombinant culture cell lines and the like. There
are no particular limitations on the cell origins, and examples include mammals such
as humans, chimpanzees, monkeys, cows, horses, pigs, dogs, cats, rabbits, rats, mice
and hamsters; and birds such as chickens. Further, cells produced by hybridization
of two or more different types of cells may also be used.
[0126] There are no particular limitations on the organ or tissue from which the cells are
derived, and examples include the blood and lymphatic system, vascular system, cranial
nervous system, bone marrow, muscle tissue, thymus gland, salivary gland, oral cavity,
esophagus, stomach, liver, gallbladder, spleen, small intestine, large intestine,
rectum, skin, cornea, lungs, thyroid, mammary gland, uterus, cervix, ovaries, testes,
pancreas, kidneys, adrenal cortex, bladder, placenta, umbilical cord, embryo, fetus,
tail, mesenchymal stem cells, and cancer cells and the like.
[0127] Furthermore, stem cells can be used favorably as the cells to be cultured. Examples
include stem cells having pluripotency such as embryonic stem cells (ES cells), induced
pluripotent stem cells (iPS cells), embryonic carcinoma cells (EC cells), embryonic
germ cells (EG cells), nuclear transfer ES cells and somatic cell-derived ES cells;
tissue stem cells such as hematopoietic stem cells, bone marrow-derived mesenchymal
stem cells, adipose tissue-derived mesenchymal stem cells, umbilical cord-derived
mesenchymal stem cells, stem cells derived from other stromal tissue, Muse cells and
neural stem cells; and various types of stem cells such as precursor cells and fibroblasts
found in various tissues such as the liver, pancreas, adipose tissue, bone tissue,
cartilage tissue and nerve tissue.
[0128] The cell culturing may employ the types of conditions typically used for the culturing
of target cells, with the specific conditions selected in accordance with the type
of cells being cultured.
[0129] There are no particular limitations on the medium used for the cell culture, provided
the cells can survive and proliferate, and the medium may be selected appropriately
in accordance with the type of cells being cultured.
[0130] Either serum media or serum-free media may be used as the medium, but the cell scaffold
material according to one of embodiments can be used favorably for culturing in a
serum-free medium.
[0131] Examples of serum-free media include Eagle Medium, Dulbecco's Modified Eagle Medium
(low-glucose or high-glucose), Eagle MEM Medium, αMEM Medium, IMDM Medium, Ham's F10
Medium, Ham's F12 Medium, RPMI-1640 Medium, and blended media of these media.
[0132] Serum media can be prepared by adding a serum to a serum-free medium. In those cases
where a serum is added to a serum-free medium, serums such as Fetal bovine serum (FBS),
horse serum and human serum may be used. When a serum is added, the serum concentration
is preferably not higher than 30%.
[0133] Additives may be added to the medium if necessary. Examples of these additives include
vitamins such as vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin
C and vitamin D; coenzymes such as folic acid; amino acids such as glycine, alanine,
arginine, asparagine, glutamine, isoleucine and leucine; sugars and organic acids
that acts as carbon sources such as lactic acid; growth factors such as EGF, FGF,
PFGF and TGF-β; interleukins such as IL-1 and IL-6; cytokines such as TNF-α, TNF-β
and leptin; metal transporters such as transferrin; metal ions such as iron ions,
selenium ions and zinc ions; SH reagents such as β-mercaptoethanol and glutathione;
and proteins such as albumin.
[0134] There are no particular limitations on the cell culture method, and a method suited
to the particular target cell may be used. The cell culture may be conducted within
a temperature range from 30 to 40°C, and preferably at a temperature of 37°C, at a
humidity within a range from 70 to 100%, and preferably within a range from 95 to
100%, and in an atmosphere containing 2% to 7% CO
2, and preferably 5% CO
2. There are no particular limitations on the cell subculture timing or method, and
a method suited to the target cell may be used.
[0135] The culture configuration may be either a two-dimensional culture in which cell adhesion
is easier, or a three-dimensional culture in which the cells are suspended in a culture
system. The culture configuration may be selected appropriately in accordance with
the type of cell and the medium configuration and the like.
[0136] Further, according to one of embodiments, a cell culture kit containing the cell
scaffold material according to one of embodiments described above, a substrate and
a medium may be provided. In this kit, the cell scaffold material according to one
of embodiments, the substrate and the medium may each be stored in a separate container.
Furthermore, by using a cell scaffold support of one of embodiments containing a substrate
and a cell scaffold material, the cell culture support and the medium may each be
stored in a separate container. Furthermore, these kits may also include the cells
to be cultured. Further, these kits may also include implements or the like to be
used in the culturing process. The cell scaffold material and cell scaffold material
support according to one of embodiments are as described above.
[0137] Further, one of embodiments can provide use of a copolymer having a polylactic acid
structural unit (A) and a polycarbonate structural unit (B) for a cell scaffold material.
[0138] Furthermore, one of embodiments can provide use of a copolymer having a polylactic
acid structural unit (A) and a polycarbonate structural unit (B) for the production
of a cell scaffold material.
[0139] Here, details relating to the copolymer having a polylactic acid structural unit
(A) and a polycarbonate structural unit (B) are as disclosed in the items described
above relating to the copolymer in the cell scaffold material according to one of
embodiments described above. For example, in the copolymer, the polycarbonate structural
unit (B) may include at least one unit having an alkoxyalkyloxycarbonyl group as a
substituent, or the polycarbonate structural unit (B) may include at least one unit
represented by general formula (I) shown above. Furthermore, in the copolymer, the
polylactic acid structural unit (A) may be a poly-D-lactic acid structural unit or
a poly-L-lactic acid structural unit. Moreover, the copolymer may be an ABA-type block
copolymer. An arbitrary combination of these items is also possible. In one example,
in the copolymer, the polycarbonate structural unit (B) either includes at least one
unit having an alkoxyalkyloxycarbonyl group as a substituent, or includes at least
one unit represented by general formula (I) shown above, and the polylactic acid structural
unit (A) is a poly-D-lactic acid structural unit or a poly-L-lactic acid structural
unit. The copolymer of this example may be an ABA-type block copolymer.
EXAMPLES
[0140] The present disclosure is described below in further detail using a series of examples,
but the present disclosure is not limited to these examples.
[Methods for Evaluating Physical Properties]
[0141] The molecular weight, structural confirmation, and degree of polymerization progression
for each of the products obtained in the examples were evaluated using the following
procedures.
(1) Molecular Weight
[0142] The number average molecular weight (Mn) and the weight average molecular weight
(Mw) were measured by connecting GPC columns (Gelpack GL-R420, R430, R440, manufactured
by Hitachi Chemical Techno Service, Ltd.) to a Chromaster GPC analysis system (manufactured
by Hitachi High-Tech Science Corporation), and eluting the sample at 1.75 mL/min using
THF as the eluent.
(2) Molecular Weight Distribution (Mw/Mn)
[0143] The molecular weight distribution was determined as the ratio (Mw/Mn) between the
values for the weight average molecular weight (Mw) and the number average molecular
weight (Mn) determined using the method described above in (1).
(3) NMR Measurement
[0144] Structural analyses of the monomers and polymers were conducted by
1H-NMR measurements using an NMR measurement apparatus (JEOL 500 MHz JNM-ECX, manufactured
by JEOL Ltd.). Chemical shifts were recorded relative to CDCl
3 (
1H: 7.26 ppm).
[Reagents]
[0145] The reagents 2,2-bis(methylol)propionic acid (bis-MPA: 98%), 2-methoxyethanol (99.8%)
and Amberlyst-15 (a registered trademark) (dry, moisture ≤ 1.5%) were procured from
Sigma-Aldrich Japan KK, and used without modification. Ethyl acetate (99.5%), dichloromethane
(DCM: 99.5%), pyridine (99.5%), ammonium chloride (98.5%), sodium bicarbonate (99.5
to 100.3%), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU: 99.0%), benzoic acid (99.5%),
diethyl ether (99.0%), hexane (95.0%), trimethylene carbonate (TMC: 98.0%), methanol
(99.8%), tetrahydrofuran (THF: 99.5%), (+)-sparteine (Sp), and 2-propanol (99.7%)
were procured from Kanto Chemical Co., Inc. Anhydrous grade THF and DCM (water content
< 10 ppm) were supplied from a solvent supply system from Kanto Chemical Co., Inc.
Hydrochloric acid (35.0 to 37.0% by mass) and magnesium sulfate (95.0%) were procured
from FUJIFILM Wako Pure Chemical Corporation, and triphosgene (98%), benzyl alcohol,
2-methoxyethyl acrylate (>98.0%), 1,4-dioxane (>99.0%) and 2,2-azobis(isobutyronitrile)
(AIBN, >98.0%) were procured from Tokyo Chemical Industry Co., Ltd., and each compound
was used without modification. Further, 1-(3,5-bis(trifluoromethyl)phenyl)-3-cyclohexyl-2-thiourea
(TU) was synthesized with reference to the method previously disclosed. The monomers
and TU were dissolved in THF and dried using CaH
2 (calcium hydride). The DBU was subjected to distillation under reduced pressure using
CaH
2 prior to use.
[0146] The D-lactic acid lactide (D-lactide) was procured from Purac NV.
[0147] Lipidure (a registered trademark)-CM5206 (hereafter also abbreviated as PMB) was
procured from NOF Corporation.
[Synthesis Example 1: Synthesis of Polylactic Acid-Polycarbonate Block Copolymer (PDMED)]
<Synthesis of 2-methoxyethyl 2,2-bis(hydroxymethyl)propanoate (ME-MPA)>
[0148]
![](https://data.epo.org/publication-server/image?imagePath=2022/18/DOC/EPNWA1/EP20832378NWA1/imgb0006)
[0149] First, bis-MPA (30.0 g, 0.224 mol) and an ion exchange resin Amberlyst-15 (a registered
trademark) (6.00 g) were added to 2-methoxyethanol (300 mL, 3.82 mol), and the resulting
mixture was stirred under heating at 90°C for 48 hours. Subsequently, the ion exchange
resin was removed from the reaction solution by filtration, and the obtained filtrate
was concentrated under reduced pressure. The product was then dried under vacuum to
obtain ME-MPA as a light yellow oily substance (41.4 g, yield: 96.3%).
1H-NMR (400 MHz, CDCl
3): δ 4.35 (t, J=4.8 Hz, 2H), 3.85 (d, J=12 Hz, 2H), 3.73 (d, J=12 Hz, 2H), 3.63 (t,
J=5.0 Hz, 2H), 3.39 (s, 3H), 1.11 (s, 3H)
<Synthesis of 2-(2-methoxyethyloxycarbonyl)-2-methyltrimethylene carbonate (MEMTC)>
[0150]
![](https://data.epo.org/publication-server/image?imagePath=2022/18/DOC/EPNWA1/EP20832378NWA1/imgb0008)
[0151] ME-MPA (20.0 g, 0.104 mol) and pyridine (50.5 mL, 0.624 mol) were added to dichloromethane
(DCM) (120 mL), and the resulting mixture was cooled to -75°C in a dry ice-2-propanol
(IPA) bath. Subsequently, a DCM solution (160 mL) of triphosgene (15.4 g, 0.0520 mol)
was added dropwise to the reaction mixture, and the resulting mixture was then stirred
under cooling at -75°C for one hour, and then at room temperature for a further two
hours. Following completion of the reaction, a saturated aqueous solution of ammonium
chloride (200 mL) was added and stirred for 30 minutes, and the organic phase was
then washed twice with a 1 N aqueous solution of hydrochloric acid (200 mL), with
a saturated aqueous solution of sodium bicarbonate (200 mL), with a saturated saline
solution (200 mL), and finally with ion-exchanged water (200 mL). The thus obtained
organic phase was dried over magnesium sulfate, and then concentrated and dried under
reduced pressure. Subsequently, the product was purified by column chromatography
(ethyl acetate), yielding MEMTC as a colorless viscous liquid (14.1 g, yield: 46.2%).
[0152] 1H-NMR (400 MHz, CDCl
3): δ 4.68 (d, J=11 Hz, 2H), 4.32 (t, J=9.5 Hz, 2H), 4.20 (d, J=11 Hz, 2H), 3.57 (t,
j=4.8 Hz, 2H), 3.33 (s, 3H), 1.31 (s, 3H)
<Synthesis of poly[2-(2-methoxyethyloxycarbonyl)-2-methyltrimethylene carbonate] (PMEMTC)>
[0153]
![](https://data.epo.org/publication-server/image?imagePath=2022/18/DOC/EPNWA1/EP20832378NWA1/imgb0010)
[0154] In a glovebox under a nitrogen atmosphere, MEMTC (0.433 g, 1.99 mmol), TU (15.0 mg,
0.041 mmol) and DBU (6.1 mg, 0.040 mmol) were stirred in DCM (2 mL) at room temperature
. After two hours of reaction, consumption of the monomers was confirmed by
1H-NMR, a few drops of benzoic acid were added as a terminator, and the mixture was
stirred overnight. Subsequently, the reaction solution was re-precipitated in 2-propanol
(60 mL), and the product was dried under vacuum to obtain a colorless, viscous polymer
PMEMTC (0.325 g, yield: 75.1%).
GPC: Mn 8,700, Mw/Mn 1.11
1H-NMR (400 MHz, CDCl3): δ 4.30 (m, 6H), 3.58 (t, J=4.8 Hz, 2H), 3.36 (s, 3H), 1.27 (s, 3H)
<Synthesis of PDLA-PMEMTC-PDLA (PDMED)>
[0155]
![](https://data.epo.org/publication-server/image?imagePath=2022/18/DOC/EPNWA1/EP20832378NWA1/imgb0011)
[0156] In a glovebox under a nitrogen atmosphere, PMEMTC (0.16 g, 0.734 mmol) was dissolved
in DCM (480 mg), a small amount of calcium hydride was added as a desiccant, and the
resulting mixture was stirred for one hour. Subsequently, the mixture was filtered
through a syringe filter, and the filtrate was added dropwise to a vial containing
(+)-sparteine (Sp) (3.44 mg, 1.49 mmol). In a separate preparation, TU (5.8 mg, 1.49
mmol) and D-lactide (DLA, 77.1 mg, 0.535 mmol) were dissolved in DCM (240 mg), and
the resulting solution was then mixed with the initially prepared solution and stirred
at room temperature. After two hours of reaction, consumption of the monomers was
confirmed by
1H-NMR, a few drops of benzoic acid were added as a terminator, and the mixture was
stirred for several hours. Subsequently, the reaction solution was re-precipitated
in 2-propanol (30 mL), and the product was dried under vacuum to obtain a white solid
PDLA-PMEMTC-PDLA (PDMED) (0.132 g, yield: 55%).
GPC: Mn 18,000, Mw/Mn 1.10
1H-NMR (400 MHz, CDCl3): δ 5.26 to 5.12 (q, 2H), 4.30 (m, 6H), 3.58 (t, J=4.8 Hz, 2H), 3.36 (s, 3H), 1.70
to 1.50 (d, 6H), 1.27 (s, 3H)
[Synthesis Example 2: Synthesis of Polylactic Acid (PDLA)]
[0157] TU (5.8 mg, 1.49 mmol) and D-lactide (77.1 mg, 0.535 mmol) were dissolved in DCM
(240 mg), and the resulting solution was stirred at room temperature. After two hours
of reaction, the reaction solution was re-precipitated in 2-propanol (30 mL), and
the product was dried under vacuum to obtain a white solid PDLA.
[Synthesis Example 3: Synthesis of PTMC (polytrimethylene carbonate)]
[0158] Benzyl alcohol (4.32 mg, 0.04 mmol), trimethylene carbonate (2.01 g, 20 mmol), DBU
(182.4 mg, 1.4 mmol) and TU (447.1 mg, 1.4 mmol) were dissolved in DCM (20 mL) and
the solution was stirred. After 24 hours, benzoic acid was added to halt the reaction,
the reaction solution was re-precipitated in 2-propanol, and the supernatant liquid
was removed by centrifugal separation. The thus obtained viscous product was recovered
by dissolution in DCM, and then concentrated and dried using an evaporator, thus obtaining
PTMC.
[Synthesis Example 4: Synthesis of PMEA (poly(2-methoxyethyl)acrylate)]
[0159] First, 15 g of 2-methoxyethyl acrylate (130.14 g/mol, 115 mmol) was dissolved in
60 g of 1,4-dioxane, and the solution was subjected to nitrogen bubbling for 30 minutes.
Next, 15 mg of AIBN (0.091 mmol) as an initiator was dissolved in a small amount of
1,4-dioxane and added to the reaction solution, and a polymerization was conducted
at 75°C for 6 hours while nitrogen bubbling was continued. Subsequently, the polymer
produced in the polymerization was collected as a precipitate by adding the polymerization
solvent medium dropwise to 1,000 ml of n-hexane. The obtained by-product was purified
by repeating the precipitation operation three times using a THF/n-hexane system.
The purified polymer was recovered by dissolution in THF, concentrated using an evaporator,
and then dried under vacuum at 60°C for 30 hours, thus obtaining PMEA.
[Methods for Evaluating Cell Cultures]
[0160] The evaluations described below were conducted using the PDMED obtained in Synthesis
Example 1, the PMEMTC that represents an intermediate in Synthesis Example 1, the
PDLA obtained in Synthesis Example 2, the PTMC obtained in Synthesis Example 3, the
PMEA obtained in Synthesis Example 4, and PMB (LIPIDURE (a registered trademark) CM5206,
manufactured by NOF Corporation) as polymers.
(1) Preparation of Cell Scaffold Material
[0161] A PET film (Diafoil T100E125 E07, manufactured by Mitsubishi Chemical Corporation)
of thickness 125 µm that had been punched out into a circular shape with a diameter
of 15 mm was subjected to a degreasing treatment by immersion overnight in methanol.
Subsequently, each polymer was dissolved in either DCM or methanol and adjusted to
a concentration of 0.2% by mass. Next, 100 µL samples of this polymer solution were
applied twice to the treated PET film using a spin coater, and following standing
for one day at room temperature, the coated film was washed by immersion overnight
in pure water. Following drying, the polymer-coated PET film (hereafter referred to
as a support) was sterilized, inside a clean bench, by irradiation for 10 minutes
with a spot UV irradiation device (SP-11, manufactured by Ushio Inc.) at an output
of 4 W/cm
2. Further, an untreated support (PET support) was prepared as a control, by conducting
the degreasing treatment, but then not coating the PET film with a polymer.
(2) Model Protein Adsorption Test
[0162] A PET film (Diafoil T100E125 E07, manufactured by Mitsubishi Chemical Corporation)
of thickness 125 µm that had been punched out into a circular shape with a diameter
of 15 mm was subjected to a degreasing treatment by immersion overnight in methanol.
Next, 100 µL samples of a solution prepared by dissolving 0.1% by mass BSA (bovine
serum albumin) in a phosphate buffer solution (PBS, pH 7.4) containing 1% by mass
of Triton X-100 were applied twice to the back surface of the substrate using a spin
coater, and following standing for one day at room temperature, the coated film was
washed by immersion overnight in pure water and then dried, thus preventing non-specific
adsorption to the PET. Subsequently, each polymer was dissolved in either DCM or methanol
and adjusted to a concentration of 0.2% by mass. Next, 100 µL samples of this polymer
solution were applied twice to the top surface of the treated PET film using a spin
coater, and following standing for one day at room temperature, the coated film was
washed by immersion overnight in pure water.
[0163] The thus obtained support was placed in a 24-well plate designed for cell culturing
(Coster 24 wells, manufactured by Corning Inc.), a fluorescent-labeled BSA (FITC labeling)
was dissolved in 0.1 w/v% PBS and added to each well in an amount of 500 µL/well,
and the well plate was shaken (tilt angle: 6°, 30 r/min) at 37°C for 60 minutes. An
untreated support (PET support) that had not been coated with the polymer was used
as a control, and was treated in the same manner as above.
[0164] Following shaking, the cells were washed with PBS, the state of adsorption was observed
using a fluorescence microscope (BZ-X, manufactured by Keyence Corporation), and the
amount of adsorption was evaluated from the fluorescent intensity. The results are
shown in FIG. 1. FIG. 1 is a graph illustrating the relative fluorescent intensity
of the fluorescent-labeled BSA when supports coated with each of the various polymers
were used, using the fluorescent intensity of the PET support as a standard.
(3) Cell Culture Tests
(3-1) Normal Human Fibroblasts
[0165] The PDMED support, PMB support and PET support prepared using the above (1) Preparation
of Cell Scaffold Material were set on the bottom surface of each well of a 24-well
tissue culture plate. Further, a well containing no set support was also prepared.
Each well was inoculated with normal human dermal fibroblasts (NHDF) at a seeding
density of 5×10
3 cells/cm
2. A medium prepared by adding 10% fetal bovine serum (FBS) to Eagle MEM medium was
added to each well.
[0166] This plate was cultured at 37°C under a 5% CO
2 atmosphere. One hour, one day, two days, three days and seven days after starting
the cell culture, the state of the cells were inspected, and at each time, 1,000 µL
of the culture supernatant was extracted and subjected to centrifugal separation at
7,000 rpm for one minute, with 500 µL of the resulting supernatant then being extracted
and supplied to a quantitative evaluation for FGF-2 (fibroblast growth factor 2).
(i) Method for Evaluating Cell Proliferation
[0167] Using a fluorescence difference microscope (MVX10, manufactured by Olympus Corporation),
the state of cell proliferation was observed, and the number of cells was measured.
FIG. 2 is a series of photographs viewed using the fluorescence difference microscope,
showing the states (a) one hour after the start of cell culturing, (b) one day after
the start of cell culturing, (c) two days after the start of cell culturing, (d) three
days after the start of cell culturing, and (e) seven days after the start of cell
culturing. FIG. 3 is a graph showing the number of cells relative to the cell culture
time.
(ii) Method for Evaluating Cell Differentiation Suppression
[0168] The culture supernatants sampled at the various cell culture times were each measured
using a human FGF-2 measurement ELISA kit (manufactured by R&D Systems, Inc.). FIG.
4 is a graph showing the FGF-2 concentration relative to the cell culture time.
[0169] Based on the cell observations conducted at each of the culture times, it was evident
that although the numbers of cells in the plate using PDMED (hereafter referred to
as PDMED wells) were lower than the plate using PET (hereafter referred to as the
PET wells) that was used as a blank, in general, a favorable level of proliferation
was maintained. In contrast, in the case of the plate using PMB (hereafter referred
to as PMB wells), it was evident that cells did not adhere, and the polymer had almost
no contribution to cell proliferation. When the state of the cells was observed, cell
proliferation with considerable spreading was observed in the PET wells, and when
the cell density increased, proliferation of cell masses was observed, which is a
feature of fibroblast proliferation. In the PDMED wells, cell spreading was minimal,
and even when the cell density increased, it was confirmed that the cells proliferated
while remaining adhered to the support, without forming masses.
[0170] Based on the FGF-2 quantitative results, it was evident that in the PMB wells in
which the cells were not adhered to the support, a large amount of FGF-2 was released
in the initial stages of the culture. In the PET wells, there was considerable fluctuation
in the release of the factor depending on the state of proliferation, and in particular,
on the seventh day, release of the factor had stopped due to mass formation. On the
other hand, it was evident that the PDMED wells exhibited a stable level of factor
release throughout the culture.
[0171] Observations of the cell proliferation also suggested that in the base of PDMED,
only cell proliferation was occurring preferentially, with the proliferation of cell
masses that represents a function of human fibroblasts not observed. This is also
clear from the stable release of FGF-2.
(3-2) iPS Cells
[0172] Using the same method as that described above in (1) Preparation of Cell Scaffold
Material, a PDMED support, PMB support, an untreated PET support as a control, and
a laminin (iMatrix 511, manufactured by Nippi Inc.) support as a control were each
prepared with a support diameter of 50 mm. Each support was set on the bottom surface
of a 60 mm dish (3010-060, manufactured by Iwaki Co., Ltd.). Each dish (referred to
as the PDMED dish, PMB dish, PET dish, and iMatrix dish respectively) was inoculated
with feeder-free iPS cells from Kyoto University in an amount of 2.5×10
4 cells/dish.
[0173] A medium (StemFit AK02, manufactured by Ajinomoto Healthy Supply Co., Inc.) was added
to each dish, and culturing was conducted at 37°C under a 5% CO
2 atmosphere. After seven days of cell culture, an inverted microscope (CKX31SF, manufactured
by Olympus Corporation) was used to observe the state of the cells, and undifferentiated
cells were separated and quantified by FACS (flow cytometry). Further, in a similar
manner to (3-1) above, from the start of culturing through to the seventh day, samples
of the culture supernatant were extracted and supplied to IL-6 (interleukin-6) and
TNFα (tumor necrosis factor α) quantity evaluations. Each of the evaluations was conducted
using an ELISA kit (manufactured by R&D Systems, Inc.).
[0174] The undifferentiated cell rate on the seventh day of culture and the concentrations
of IL-6 and TNFα on the fifth day of culture are shown in Table 1.
[0175] An undifferentiated cell rate (%) closer to 100 (%) means better suppression of cell
differentiation. Furthermore, an IL-6 concentration closer to 0 means better suppression
of cell differentiation. Moreover, a TNFα concentration closer to 0 means better suppression
of cell differentiation.
[Table 1]
Item |
PET |
PMB |
PDMED |
iMatrix |
Undifferentiated cell rate (%) |
97.1 |
98.8 |
99.8 |
90.2 |
IL-6 (pg/mL) |
0.06 |
0.33 |
below detection limit |
9.58 |
TNFα (pg/mL) |
below detection limit |
below detection limit |
below detection limit |
1.20 |
[0176] IL-6 is known as a cytokine associated with differentiation induction. These test
results revealed that in the case of the iMatrix dish which acted as one control,
the undifferentiated cell rate of the viable cells was lower than the other dishes,
and the levels of IL-6 and TNFα release on the fifth day of culture were much higher
than the other dishes. Based on these results, it was evident that cell differentiation
was not adequately suppressed in the iMatrix dish.
[0177] Further, in the PMB dish and the PET dish, the undifferentiated cell rate on the
seventh day of culture was lower than that of the PDMED dish, and on the fifth day
of culture, IL-6 release was also confirmed. In the case of the PMB dish, TNFα release
of about 2 pg/mL was also confirmed on the second day of culture (data not shown).
These results indicated that even in the PMB dish and the PET dish, cell differentiation
was not adequately suppressed.
[0178] In contrast to these cases, in the PDMED dish, the undifferentiated cell rate on
the seventh day of culture was high, and no release of either cytokine was detected
from the start of culturing through to the fifth day. Based on these results, it was
evident that when PDMED was used as the cell scaffold material, a contribution to
proliferation could be achieved while suppressing cell differentiation.
[0179] The disclosure of the present disclosure is related to the subject matter disclosed
in prior
Japanese Application 2019-120033 filed on June 27, 2019, the entire content of which is incorporated by reference herein. It should be noted
that, besides those already mentioned above, many modifications and variations may
be made to the above embodiments without departing from the novel and advantageous
features of the present disclosure. Accordingly, all such modifications and variations
are intended to be included within the scope of the appended claims.